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Tools to Measure D-Amino acid signaling in the brain - Jonathan V. Sweedler

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Watching at the "D" side: D-amino acids and their significance in neurobiology
June 05 -June

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Tools to Measure D-Amino acid signaling in the brain - Jonathan V. Sweedler

  1. 1. Tools to Measure D-amino acid signaling in the brain Como Italy Jonathan V. Sweedler University of Illinois Urbana, IL USA Funding from: The UIUC / NIDA Neuroproteomics Center on Cell-Cell Signaling, DOE, NSF, NINDS, CNLM
  2. 2. Tools to Measure D-amino acid signaling in the brain  Affiliations  Professor of Chemistry, Neuroscience Program, MIP, IGB, & Beckman Institute  PI of the NIDA Neuroproteomics Center on Cell to Cell Signaling  Editor, Analytical Chemistry  Tools & methods  Metabolomics, Neuropeptidomics, Proteomics (development and applications)  Single cell neurochemical measurements (a set of unique measurements)  Animal models from corals, mollusks, arthropods to vertebrates
  3. 3. Funding from: The UIUC / NIDA Neuroproteomics Center on Cell to Cell Signaling NIH, NSF and the CNLM Per Andren Rohit Bhargava John Erdman Russ Fernald John Gerlt Martha Gillette Rhanor Gillette William Gilly Joshua Gully Paul Gold Neil Kelleher The Sweedler group (2010, 2014 and 2015) Jean Pierre Mothet Leonid Moroz Phil Newmark Amydah Pradham Justin Rhodes Sandra L. Rodriguez-Zas Gene Robinson John Rogers Taher Saif Klaude Weiss Mark Wightman Collaborators
  4. 4. Ouline  Approaches for D-Amino Acids  Liquid Chromatography  Capillary Electrophoresis  Microfluidics  Examples of D-Asp, D-Glu and D-Ala  Approaches for D-Amino Acids in Peptides  The discovery of novel DAACPs
  5. 5. Ala Arg Asn Asp Val LeuIle Ser Thr Met Phe ProLysHis Gln Glu Trp Tyr Gly Cys allo-Thrallo-Ile HC CH3 CH3 CH3C H CH2 CH3 CH2 HC CH3 CH3CH3 CH CH2 CH3 CH3 CH2 CONH2 CH2 COOH CH2 SH (CH2)4 NH2 (CH2)2 S CH2 OH CH2 H CH2 NH (CH2)3 NH NH2CHN CH2 CH2 CONH2 CH2 CH2 COOH N CH3 CH2 HC NH COOH C H CH3 HO N CH2 OH C OH CH3 H Targets All proteinogenic amino acids Slides from Prof. Kenji Hamase, Kyushu University
  6. 6. 2D-HPLC separation of chiral AAs Auto sampler R WasteMonolithic ODS 0.53 x 1000 mm HPV RD Waste 1D Multi-loop valve P P P P P KSAACSP-001S 1.5 x 250 mm 2D D 1D: Reversed-phase separation of NBD-amino acids 2D: Enantiomer separation of amino acids ••• D L L D L D L D •••
  7. 7. 公開講演会091216 His 1D: reversed-phase separation Asn Ser Gln Arg Asp Gly allo-Thr Glu Thr Ala Pro Met Val allo-Ile Ile Leu Phe Trp Lys Cys Tyr D Asn Ser Gln GlyAspHis Arg D D D D D L L L L L L PheIle Leu Lys Cys TyrTrp D D D D D D D L L L L L L L allo-Thr Thr AlaGlu ValMet allo-IlePro D D D D D D D D L L L L L LL L 2D: enantiomer separation 2D-HPLC separation of chiral AAs
  8. 8. Separation of all proteinogenic amino acid enantiomers in the serum of a patient with chronic kidney disease L L L L L LL LL L L L L L L L L L L His Asn Ser Gln Arg Asp Gly allo-Thr Glu Thr Ala Pro Met Val allo-Ile Ile Leu Phe Trp Lys Cys Tyr D D D D L Ser D L Ala D L Pro
  9. 9. 公開講演会091216 Nat. Neurosci., 14, 603-611 (2011).
  10. 10. 公開講演会091216 Proc. Natl. Acad. Sci. USA, 109, 627-632 (2012).
  11. 11. His GlnAsn Gly allo-Thr Thr AlaGlu Val allo-Ile LeuPro Phe Trp TyrLys AspArg Ile Cys L L L L L L L L LL L L L L LLL Met Ser D D D D Enantiomer separations of NBD-amino acids in a yogurt sample using a 2D-HPLC-FL system
  12. 12. His GlnAsn AspArg Gly allo-Thr Thr AlaGlu ValMet allo-Ile Ile LeuPro Phe Trp Cys TyrLys L L L L LLL L L L L L L LL Ser D D D D D D Enantiomer separations of NBD-amino acids in a Kurozu sample using a 2D-HPLC-FL system
  13. 13. Capillary electrophoresis laser induced fluorescence system measurements from nanoliter volume samples • System is well suited for chiral separations • Has the ability to work with samples as small as an individual cell • Is not automated and has the quirks associated with most lab assembled equipment Linhardt, R.J. et al. Science, 298, 1441-1442 , (2002)
  14. 14. Capillary electrophoresis laser induced fluorescence measurements from nanoliter volume samples A lab-built CE-LIF system
  15. 15. Chiral Capillary Electrophoresis Laser-Induced Fluorescence Chiral Selector: Quaternary Ammonium Beta Cyclodextrin (QAβCD) N-substituted 1-cyanobenz[f]isoindole amino acid 440 490 15 Fluorescent Tag: Naphthalene-2,3-dicarboxaldehyde (NDA) + +-- 0 +2-2
  16. 16. Chiral Selector: β-cyclodextrin; Chiral Surfactant: Deoxycholate
  17. 17. Can we measure the chemistry at the single cell level. First, we isolate the neuron Analyte extraction 50% methanol, 0.5% acidic acid (Rinse w/ water) Aplysia californica 3. Enzymatic treatment 4. Dissection (ganglia and nerves placed in artificial seawater) 1. Anesthesia 2. Sacrifice 1 mm CNS R2 neuron250 µm 250 µm Single-cell Isolation in 33% glycerol Cell culturing
  18. 18. Comparison of serotonin in a single neuron from hungry and satiated Pleurobranchaea Hatcher et al, J. Neurochemistry, 2008, 104(5):1358. 5-HT levels in a single cell tracks animal’s hunger level Fuller, Neuron 20, 1998, 173–181.
  19. 19. One can even collect the output of from CE for other follow-up experiments
  20. 20. Kennedy’s lab, University Michigan Anal. Chem., 2009, 71 (13), pp 2379–2384 Microdialysis coupled on-line with optically gated CE-LIF Rapid separation of d,l- aspartate as the OPA/BME derivative. Electropherograms obtained from adrenal gland homogenates of 3-week-old rats: Peak 1 was identified as glutamate, peak 2 as d-aspartate, and peak 3 as l- aspartate. Faster CE separations and coupling to dialysis
  21. 21. Everything changes on the other side of the mirror…. http://elusiveheroine.wordpress.com/2010/10/10/w here-have-i-been-and-where-am-i-going/ Moving to biological experiments….
  22. 22. Is D-Asp a neurotransmitter? • There are four main criteria for identifying neurotransmitters: – The chemical must be synthesized in the neuron or otherwise be present in it. – When the neuron is active, the chemical must be released and produce a response in some target. – The same response must be obtained when the chemical is experimentally placed on the target. – A mechanism must exist for removing the chemical from its site of activation after its work is done. • However, more recently the term "neurotransmitter" can be applied to chemicals that: – Carry messages between neurons via influence on the postsynaptic membrane. – Have little or no effect on membrane voltage, but have a common carrying function such as changing the structure of the synapse. – Communicate by sending reverse-direction messages that have an impact on the release or reuptake of transmitters. • NOTE: to determine this, single cell measurements are required! From Wikipedia: 2/29/016
  23. 23. Molluscan model system (Aplysia)  Relatively simple sensory and motor organs (the head and the tail)  All basic classes of behavior: foraging, predator avoidance and reproduction What is this, why was it on the cover of the Journal of Neuroscience?
  24. 24. Advantageous features of its CNS  Neuronal cell bodies (& neuropile): discrete ganglia, each has preferential functions (Kandel 1979)  Axonal bundles: connectives; nerves  Circuits for behavior with small number of identifiable neurons: a direct link of their roles in behavior; can we link novel neurochemisty to behavior? Cerebral ganglia Buccal ganglia Pedal ganglia Ring ganglia Abdominal ganglionCourtesy of Jian Jing, Nanjing Univeristy
  25. 25. Circuit analysis and approaches Intracellular Extracellular Courtesy of Jian Jing
  26. 26. D-asp percentage (%) in identified clusters of cerebral ganglia 0 10 20 30 40 50 60 70 80 90 100 F-cluster C-cluster G-cluster (D-asp)/(totalAsp)x100% Isolate the sample containing the neurons, separate them with capillary electrophoresis, and quantify the D-Asp/L-Asp using CE-LIF with NDA derivatization and MEKC n=6 n=6 n=6 Does Aplysia have D-Asp? Analysis of plysia neuronal clusters in the cerebral ganglion
  27. 27. Time 800 1000 1200 1400 1600 Tau D-Asp I.S. Glu* L-Asp Tau Glu* D-Asp L-Asp I.S. A B FluorescenceIntensity Neurochemical measurements from a single process 100 mm Cell body Neurite R2 Neuron from Abdominal Ganglion
  28. 28. D-Asp in single sensory neuron Time (sec) 1000 1200 1400 1600 1800 D-Asp I.S. L-Asp L-Leu L-Phe D-Asp L-Asp L-Leu L-Phe I.S. Unit Scale Unit Scale FluorescenceIntensity Single Cell Processes Single cell soma Single Sensory Neuron from Pleural Ganglion 30 µM Miao et al. J. Neurochem. 2006, 97, 595.
  29. 29. Incorporate Radiolabels Is D-Asp created from L-Asp? -+ L-Asp Isolate hemiganglia Incubate 24 hrs Separation of D,L-Asp by CE Derivatize 1. CE-LIF experiment -+ -L-Asp Isolate ganglion Incubate 24 hrs Separation of -D-Asp, -L-Asp by CE Derivatize 2. Radiolabel experiment
  30. 30. L- to D-Asp Conversion occurs predominantly in the cerebral ganglia neurons. No significant D- to L-Asp conversion occurs in the cerebral ganglion. Desheathed area Sheath Using radioactivity do measure conversion of L- to D-Asp and D- to L-Asp Scanlan et al. Neurochem. 115, 2010,1234
  31. 31. Uptake of 14C-Asp in single cells after 20 hours incubation 0.00E+00 5.00E-12 1.00E-11 1.50E-11 2.00E-11 2.50E-11 3.00E-11 3.50E-11 F-cluster cell (D) B-cluster neuron (D) F-cluster cell (L) B-cluster neuron (L) moles n=8 n=5 n=7 n=7 Preference for 14C-D-Asp uptake observed in both sets of cells Selective uptake of D-Asp in single neurons
  32. 32. Is D-Asp a hormone? CE-LIF Analysis of Release into blood (A) Prestimulation - Both D and L-Asp observed (B) Stimulation by 10 µm Ionomycin - Only D-Asp collected (~150 pmols!) (C) Spiked with D- and L-Asp - L-Asp peak forms at correct location Ionomycin stimulation increases D-Asp release from F-cluster cells Place SPE beads on AT and UL nerve and measure releasate
  33. 33. D-Asp release from the entire cerebral ganglion Scanlan et al. Neurochem. 115, 2010,1234
  34. 34. Electrophysiological Stimulation by D-Asp D-Asp puff (3 mM) stimulation of abdominal ganglion stimulates action potential bursting. (A) Recording from PAC nerve - D-Asp has more pronounced effect (B) Recording from Single R2 Cell - Some initial hyperpolarization by D-Asp but not L-Asp
  35. 35. Extracellular applications of D-Asp to distinct regions of neurons produce different responses Cultured Aplysia neuron Intracellular recording electrode Application pipette Application pipette 10 mM D- or L-Amino acid Schematics of extracellular AA application experiments 3 -2.5 -2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 Depolarizing effect of D-Aspartate D-Asp L-Asp D-Asp L-Asp Cell body Terminals Changeofmembrane potential(mV) Changeofmembrane potential(mV) Hyperpolarizing effect of D-Aspartate D-Asp L-Asp D-Asp L-Asp Cell body Terminals Mean ± SE 20 s 1 mV TerminalD-Asp 40 s 2 mV Cell bodyD-Asp n=2 n=2 Sweedler group unpublished
  36. 36. D-Asp Stimulates Peptide Release from an identified neuron • D-Asp puff (3 mM) stimulation of abdominal ganglion stimulates secretion of R3-14 peptides. - (A) Prestimulation - (B) Stimulation • No other peptides detected after application of either D- or L-Asp. Sweedler group unpublished
  37. 37. How is it made? Cloning and expression of a neuronal d-Asp racemase Dehydratase activity  Bioinformatics to identify putative racemases  Cloned most likely enzymes  Expressed in E coli and purified  Testing activity and localization Converts L-Asp to D-Asp Known racemases also have Serine dehydratase activity Wang et al, JBC, 2011
  38. 38. always conserved residues; identical residues; similar residues; different residues Aplysia d-Asp racemase
  39. 39. Unique dual racemase activity (measured via CE-LIF) Wang et al, JBC, 2011
  40. 40. BG PdG PlG CG C, F, F,C 500μm 500μm 500μm Enzyme localization in Aplysia matches the cells where we had previously found D-Asp CNS whole mount Cerebral ganglia
  41. 41. Enzyme colocalizes with d-Asp: what about d-Ser? Different CE-LIF conditions required to separate the two enantiomers
  42. 42. Biosynthesis of D-Asp and D-Ser in Aplysia Wang, L. P. et. al. J. Biol. Chem. 2011, 286, 13765-13774. F- and C-clusters of cerebral ganglia
  43. 43.  D-Asp is localized to specific neurons  D-Asp is synthesized in those neurons  D-Asp is released in a Ca2+-dependent and activity-dependent manner  D-Asp has Na+-dependent uptake  D-Asp, as L-Asp, causes electrical activity in post-synaptic cells  D-Asp for some cells is sufficient to cause peptide release Evidence is consistent with D-Asp as a neurotransmitter and hormone
  44. 44. D-Glu: found throughout the Metazoan A. Mangas et al., Neuroscience, 144, 19 January 2007, Pages 654-664 Using a specific D-Glu antibody, performed an immunocytochemical visualization of d-glutamate in the rat brain; using other selective antibodies, no d-tryptophan-, d- cysteine-, d-tyrosine- and d- methionine-immunoreactive structures were found. No functional tests have been performed. Interestingly, d-Asp- oxidase works with both d-Glu and d- Asp.
  45. 45. 25 26 27 28 29 30 0 100000 200000 300000 400000 500000 FluorescenceIntensity(RFU) Time (min) Analytical Challenges of D-Glu Quantitation L-Glu D-Glu L-Asp α-CD in borate buffer D-Asp 32 33 34 35 36 37 38 0 1000000 2000000 3000000 4000000 5000000 FluorescenceIntensity(RFU) Time (min) α-CD/camphor sulfonate/ quinine In formamide L-Glu D-Glu L-Asp D-Asp Confident D-Glu quantitation requires well-separated D-Glu peak 0 5 10 15 20 25 30 0.0 1.0x10 7 2.0x10 7 3.0x10 7 4.0x10 7 5.0x10 7 FluorescenceIntensity(RFU) Time (min) D-Glu L-Glu 24.0 24.5 25.0 25.5 26.0 F- and C-clusters of cerebral ganglia
  46. 46. Large Volume Sample Stacking Fill entire capillary with low conductivity sample Enantiomer 1 Enantiomer 2 QAβCD 46
  47. 47. Large Volume Sample Stacking Apply fixed voltage Analytes stack at low conductivity-high conductivity boundary EOF 47
  48. 48. Large Volume Sample Stacking Apply fixed voltage Analytes stack at low conductivity-high conductivity boundary EOF 48
  49. 49. Large Volume Sample Stacking Apply fixed voltage Analytes stack at low conductivity-high conductivity boundary EOF 49
  50. 50. Apply fixed voltage Analytes stack at low conductivity-high conductivity boundary Large Volume Sample Stacking EOF 50
  51. 51. Large Volume Sample Stacking EOF 51
  52. 52. Sample matrix forced out of capillary inlet Analyte band reverses direction Large Volume Sample Stacking EOF 52
  53. 53. Sample matrix forced out of capillary inlet Analyte band reverses direction Large Volume Sample Stacking EOF 53
  54. 54. Sample matrix forced out of capillary inlet Analyte band reverses direction Large Volume Sample Stacking EOF 54
  55. 55. Sample matrix forced out of capillary inlet Analyte band reverses direction Large Volume Sample Stacking EOF 55
  56. 56. Large Volume Sample Stacking Standard CE separation with concentrated sample band EOF 56
  57. 57. Large Volume Sample Stacking Standard CE separation with concentrated sample band EOF 57
  58. 58. Standard CE separation with concentrated sample band Large Volume Sample Stacking EOF 58
  59. 59. 59 Example Electropherogram L-Asp D- Asp D-Glu L-Glu D-Glu
  60. 60. D-Asp Analysis in Cerebral Ganglia: Relative Amounts 60 Analysis of pleural sensory neurons, pleural PL1, abdominal L11/L7, and cerebral MCC all detected 10-40% D-Asp ♦ = Mean from 3 technical replicates □ = Mean of Dataset Error Bars = Range of Dataset Lower line: 1st quantile Middle line: Median of Dataset Upper line: 3rd quantile
  61. 61. D-Glu Analysis in Cerebral Ganglia: Relative Amounts 61 Analysis of pleural sensory neurons, pleural PL1, abdominal L11/L7, and cerebral MCC all resulted in no detected D-Glu ♦ = Mean from 3 technical replicates □ = Mean of Dataset Error Bars = Range of Dataset Lower line: 1st quantile Middle line: Median of Dataset Upper line: 3rd quantile
  62. 62. D-AA Analysis in Cerebral Ganglia: Relative D-Asp and D-Glu Comparison No apparent correlation between D-Asp and D-Glu in studied cell types Is an enzyme other than DAR1 or DAR1L responsible? 62 DAR1L reported by Uda et al. Amino Acids, 2016, 48 (2), 387-402
  63. 63. D-AA Analysis in Cerebral Ganglia: Absolute Amounts 63 Quantitative analysis by standard additions (in triplicates) Note the presence of solely the L-forms in the sample blank (cell isolation media)
  64. 64. Lets go primitive and look at the comb jelly Moroz et al, Nature 2014 (510), 109.
  65. 65. Glu as Neuromuscular Transmitter in Pleurobrachia Two Neural Systems in Ctenophores a b GABA Glu ACh Histamine InnexinExpression * c Normalized stimulus intensity’ d Polar Fields ‘Triad’ Synapses ‘Classical’ Synapses ‘Elementary Brain’
  66. 66. Detection of glutamate & aspartate enantiomers in Ctenophores
  67. 67. The most primitive neuromuscular transmitters L- Glu (and L/D-Asp and D-Glu) induced action potentials & contractions in muscle cells GABA-IR muscles
  68. 68. While d/l Asp/Glu are detected and bioactive, we do not detect indoles and catecholes in the Ctenophores but do throughout the rest of the Metazoan
  69. 69. Optimized chiral separation of standard CBI-amino acids by NACE. The concentration of each amino acid is 1 µM and the FL concentration is 2 µM. The separation buffer contained 40 mM HP-γ-CD, 80 mM CS, 80 mM QN, and 10 mM sodium acetate in FA; separation was performed at 27 kV normal polarity over 40 cm of bare fused- silica capillary (75 µm inner diameter) and a detection window at 30 cm from the inlet. Another example: D-Ala in the mammalian endocrine system Ota et al, BBRC , 2014
  70. 70. (A) Electropherograms of separated Ala enantiomers just after the addition of DAAO (bottom) and after 1 h incubation at 37 °C (top). The separation conditions are the same as described in Figure 1. (B) Schematic of D-Ala oxidation by DAAO at the presence of the cofactor FAD that is regenerated by the activity of catalase. Ota et al, BBRC , 2014 Confirming the identity of a D-Ala peak using enzymatic oxidation
  71. 71. (A) Islet of Langerhans (arrow) can be visually distinguished from surrounding acinar tissue after enzymatic and mechanical treatments. Several transfers into dishes with fresh physiological solution and visual examination of collected sample ensure the quality of islet preparation. (B) Using matrix-assisted laser desorption/ionization mass spectrometry, we profile the peptides in the islets; we detect neuropeptides expected for Islets of Langerhans including glucagon and insulin-related peptides. Measuring D-Ala from the islets of Langerhans of rat pancreas: confirming our isolation
  72. 72. Electropherograms of an islet sample without DAAO treatment (bottom) and the same sample after DAAO treatment (top) are shown. D-Ala peak in the original sample disappeared after 1 h of DAAO digestion at 37°C. NACE-LIF analysis of isolated islets demonstrating presence of D-Ala
  73. 73. D-Ala in islet releasate before/after stimulation with glucose Electropherograms acquired from releasate sample (bottom trace), the same releasate sample with DAAO treatment (middle), and the same releasate sample after DAAO treatment and 1 µM CBI- D-Ala spike after derivatization (top). Red arrow heads indicate D-Ala peaks and black arrow heads point towards the region of electropherogram where D-Ala peak typically presents. Enzymatic treatment produces additional signals (e.g. peaks denoted with *). A set of numbered peaks was used as reference points of migration time to determine the location of D-Ala peak. These reference peaks were independent of DAAO treatment and bracket D-Ala peak.
  74. 74. Summary of free D-Amino acids  D-Asp fits the definition of a classical neurotransmitter in Aplysia and is found across the metazoan  D-Glu remains enigmatic, and is a sparsely distributed amino acid  D-Ala is at high levels in the anterior pituitary and islets, and in islets, it is released in a glucose- dependent manner.
  75. 75. Neuropeptides Neuropeptide processing defies prediction: need to make the measurement The effect of a specific neuropeptide depends on its exact chemical structure: dozens of enzymes are required process a prohormone to its final peptide products “It has been claimed that at least three of the seven deadly sins are mediated by neuropeptides.” Charles F. Stevens (forward for Neuropeptides, Fleur Strand, 1998)
  76. 76. The Aplysia insulin prohormone processed into insulin and other peptides Prepro-insulin contains the signal sequence pro-insulin is the prohormone without the signal sequence signal sequence J. Neurosci. 19, 1999, 7732–7741.
  77. 77. The mass spectrometer and bioinformatics: often the heart of a metabolomics/peptidomics measurement Dealing with COMPLEXITY INSTRUMENTATION Characterizing a neuropeptide: from the analytical perspective, use a good tool
  78. 78. MS-Based approaches for neuropeptide discovery Min0 20 40 mAU Species 1 Species 2 LC Purification of Homogenate ESI-MS to verify AA sequence and PTMs 400 1200 2000 Mass (m/z) x6 b16 y14 b9 y9 b8 b7 –H2O b20 – NH3 +2 b6 b5 b12 – H2O b29 + H2O+2 b9– H2O y7– H2O b14 – H2O b15 – H2O Sample Plate Single Aplysia R3-14 Neuron MALDI MS Spectrum Matrix 500 2900 5300 Mass (m/z) %Intensity 1380 4925
  79. 79. Are we missing a PTM? D-amino acid-containing peptides (DAACPs)  Peptide hormones: crustacean hyperglycemic hormone  Toxins in peripheral areas: conotoxin family, frog skin toxins family, platypus and spider venoms…  Neuropeptides: Achatin-I GdFAD Fulicin FdNEFVa Fulyal YdAEFLa NdWFa NdWFa Ocp-1/4 GdFGD/GdSWD  If PTM is present, we could miss important biologically active peptides In higher organisms, enzyme-mediated isomerization Peptidylaminoacyl-L/D-isomerases: 3 identified, 2 known sequences Different structures and active sites Different sites of isomerization Bai L., et al., Bioanal. Rev. 1, 2009, 7–24.
  80. 80. DAACPs as neuropeptides Neuropeptide Sequence Species Achatin I GdFAD Achatina fulica Fulicin FdNEFVamide Achatina fulica Fulyal YdAEFLamide Achatina fulica NdWFa NdWFamide Aplysia kurodai Ocp-1/4 GdFGD/GdSWD Octopus minor Mytilus-FFRFamide AdLAGDHFFRFamide Mytilus edulis  Isolated from nervous tissue: all bioactive except Ocp-4  A common feature is the isomerization in the second n-terminal position
  81. 81.  3D structure changes  Usually changes bioactivity  More resistant to degradation by peptidases  Different retention time on high pressure liquid chromatography (HPLC) Synthetic L-Phe13 conomarphin Endogenous conomarphin (D-Phe13) Consequences of isomerization
  82. 82. How do you detect isomerization when both peptides have the same mass?  Can not detect this change through MS alone  Traditionally required either:  Deep knowledge about peptide bioactivity  Homology to a known DAACP G dF F DG F F D The Challenge
  83. 83. Distinguish Peptide Epimers by Fragmentation Patterns  Secondary structures can be probed in the gas phase  Soft ionization allows solution phase structure to be carried into gas phase- >MS/MS  MALDI suitable for use with biological samples  Challenges:  Application to complex CNS samples not reported before  Application to 2nd-position isomerization wasn’t established  Considerations:  maximizing signal + minimizing variation Adam CM et al, J Am Soc Mass Spectrom. 2004, 15, 7, 1087-1098; Sachon E. et al, Anal. Chem. 2009, 81, 4389–4396.
  84. 84. Samples Bai, L. et al, Anal Chem. 2011 Apr 1;83(7):2794-800. Fragmentation Pattern Analysis with MALDI-TOF/TOF Experimental conditions: • 5,000 shots/acquisition • Normalize peak intensity to the sum of major fragment ions N=6 N=6
  85. 85. Abdominal ganglia IHC staining agst NdWFa IHC staining: courtesy of Dr. Ferdinand Sven Vilim Neuron isolation Peptide profiling Fragmentation Pattern Analysis with MALDI- TOF/TOF to Distinguish Peptide Epimers MS/MS Experimental conditions: • Same with synthetic peptides • Salt removal • Spot-to-spot cell transfer Bai, L. et al, Anal Chem. 2011 Apr 1;83(7):2794-800. Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18 ( 4) 151– 160
  86. 86. Fragmentation Difference Can Be Correlated with the Isomeric Content of N(d)WFa Bai, L. et al, Anal Chem. 2011 Apr 1;83(7):2794-800.; Sheeley, S. A. Et al, Analyst. 2005, 130, 1198-1203 Time (min) Capillary electrophoresis separation of NdWFa-containing neurons Intensity(abs.) N=10 ln(RM) Relationship of fragment ion ratio to isomeric content 0 20% 40% 60% 80% 100% D/(D+L) NdWFa >80% D-form in the analyzed cell
  87. 87. Tandem MS analysis of G(l/d)FFD G(l or d?)FFD RelativeIntensity G(l/d)FFD in Aplysia: is it a DAACP? Peptide profiling of isolated neurons ISH staining: courtesy of Dr. Ferdinand Sven Vilim N=3 Bai, L. et al, Journal Biological Chemistry, 2013 ISH staining for G(l/d)FFD expression
  88. 88.  Acid hydrolysis: High yield and low racemization desired  Liquid- and vapor-phase; incubation time  Short vapor-phase optimal  Capillary electrophoresis analysis of GdFFD hydrolysates  Sensitive and quantitative, suitable for specific peptides Chiral Selector: β-cyclodextrin; Chiral Surfactant: Deoxycholate
  89. 89. Characterization of GdFFD in A. californica Active in the feeding network Our data confirms GdFFD acts as a neuropeptide in the Aplysia feeding network: The first DAACP with a defined role in an identified neuron Electrophysiological recordings via Jian Jing, Nanjing University Bai, JBC, 2013
  90. 90. Confirmation L L L L L L L L L L L L L L L L L L L L L D L L Chiral Analysis L L L L L L L L L D L L Screening Next steps: non-targeted detection of DAACPs from biological samples Moroz, LL, et al, Cell 2006, 127, 1453-1467.
  91. 91. The DAACP Discovery Funnel Step 1 - Screening Confirm Screening Confirm Chiral Analysis GdFFDGFFD Split-peak elution using liquid chromatography Isomerization alters shape Resist degradation by aminopeptidase M DAACPs have a longer half-life - YdAEFLa (DAACP resists degradation) - NdWFa (resists degradation) - Angiotensin 1 (all-L-AA peptide, degraded) Intensity(103counts)
  92. 92. 1. Acid Hydrolyze the peptide DAACP Discovery Funnel Step 2 – Chiral Analysis L D L L L D L L 6M DCl 150 oC 30 min Confirm Screening Confirm Chiral Analysis 2. Derivatization with Marfey’s Reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) Bhushan R, Bruckner H, Amino Acids 2004, 27, 231-247.
  93. 93. DAACP Discovery Funnel Step 2 – Chiral Analysis Confirm Screening Confirm Chiral Analysis 3. Detection with Triple-Quadrupole MS (Bruker EvoQ)
  94. 94. Compare LC retention times Synthesized standard + endogenous peptide Bioactivity studies The DAACP Discovery Funnel Step 3 – Confirmation GdFFDGFFD Bai, L and Livnat I, et al, Journal of Biological Chemistry 2013, 288, 32837-32851. Screening Confirm Chiral Analysis
  95. 95. Other peptides in achatin-like prohormone may be DAACPs Important note: Targeted for their resistance to digestion by APM! Endogenous GdYFD 0 h APM Endogenous GdYFD 24 h APM GdYFD Other peptides
  96. 96. Chiral analysis also shows a D-Tyr in GYFD Endogenous G(l/d)YFD Labeled amino acid standards
  97. 97. GYFD is homologous to GFFD: shown to be GdYFD Endogenous G(l/d)YFD GYFD peptide std GdYFD peptide std
  98. 98. SYADSKDEESNAALSDFAED contains a D-Tyr (chiral analysis) Endogenous S(l/d)YADSKDEESNAALSDFA Labeled amino acid standards
  99. 99. Endogenous peptide matches retention time of spiked synthetic SdYADSKDEESNAALSDFAED Endogenous peptide spiked with SYADSKDEESNAALSDFA Endogenous peptide spiked with SdYADSKDEESNAALSDFA All-L-peptide DAACP This DAACP is confirmed… but not bioactive on the feeding network
  100. 100. Advantages:  Non-targeted analysis with APM  Directly assays all 19 common chiral amino acids Limitations:  Enzymatic digestions: false positives, but they may be true? CHH has pGlu AND D-amino acid  Chiral analysis: DCl hydrolysis can alter or degrade amino acids (including racemization), gives no sequence info Has worked to discover two new DAACPs from the achatin-like (GdFFD) prohormone. We have started testing in other animal models. Discovery funnel: Discussion Screening Chiral Analysis Confirm
  101. 101. Scientific American, 1988 D-amino acids and D- amino acid-containing peptides are found throughout the metazoan; their synthesis, localization and function are still being discovered. In every organism studied, unexpected and novel transmitters and peptides are still being found A particular challenge is the dynamic range of the brain, from length, time and chemical complexity...

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